Process for cultivating microorganisms on a hydrocarbon feedstock employing a carbohydrate pretreatment feedstock



it States This invention relates to the cultivation of yeast by aprocess comprising starting the growth of yeast on a carbohydratestarter feedstock followed by growth on a main feedstock containing astraight chain hydrocarbon.

According to the present invention there is provided a process whichcomprises, in a pretreatment stage, cultivating a microorganism in thepresence of a pretreatment feedstock comprising a compound containingcarbon, hydrogen and oxygen and thereafter, in a growth stage,cultivating the microorganism product so obtained in the presence of agrowth feedstock comprising a straight chain hydrocarbon.

By operating in this manner the lag phase, which usually occurs when amicroorganism is first cultivated on a hydrocarbon feedstock, is reducedin its effect or is eliminated.

It will be necessary, in both the pretreatment stage and in the growthstage to operate in the presence of a gas containing free oxygen and inthe presence of an aqueous nutrient medium. This medium may be ofdifferent composition in the two stages.

When operating in batchwise manner the two stages may be carried out ineither the same or different fermenters.

When operating in continuous manner separate fermenters or separatezones in the same fermenter will be necessary.

Usually the feedstock used in the pretreatment stage will be acarbohydrate, for example consisting of or contained in molasses, wort,malt extract, wood hydrolysis sugars or lyes obtained in the'course ofpaper manufacture. A special pretreatment feedstock which may beemployed is an extract of lipids obtained in the purification of yeastsby solvent extraction.

Preferably the hydrocarbon is C or higher. Suitably there may be used ahydrocarbon fraction derived from petroleum.

It is well-known that certain petroleum fractions, particularly gasoils, contain straight chain hydrocarbons, mainly parafiins which arewaxes and which have an adverse effect upon the pour point of thefraction; that is to say, when these hydrocarbons are removed, wholly orin part, the pour point of the fraction is lowered. Usually the wax isremoved by precipitation by means of solvents, the wax originallypresent in the fraction being recovered as such, that is, withoutconversion to more valuable products.

The petroleum fractions boiling below the gas oils, for example, heavynaphthenes and kerosines also contain straight chain hydrocarbons whichare potentially valuable for conversion to other products but hitherto,in general, utilisation of these hydrocarbons has been rendereddifficult by the necessity of recovering these hydrocarbons from thepetroleum fractions, in which they are contained, before they can beconverted to other products.

According to a preferred feature of this invention there is provided 'aprocess which comprises cultivating a microorganism in the manner ashereinbefore described in the presence of a petroleum fractionconsisting in part of atent straight chain hydrocarbons and having amean molecular weight corresponding to at least 10 carbon atoms permolecule, and in the presence of an aqueous nutrient medium; and in thepresence of a gas containing free oxygen and separating from themixture, on the one hand, the microorganism and, on the other hand, apetroleum fraction having a reduced proportion of straight chainhydrocarbons or which is free of said straight chain hydrocarbons.

The process of the invention is of particular value for the treatment ofpetroleum gas oil fractions which contain straight chain hydrocarbons inthe form of waxes, since by the process of the invention, a gas oil ofimproved pour point is obtained while the waxes are converted to avaluable product.

Usually the straight-chain hydrocarbons will be present in thefeedstocks according to the invention as paraffins; however, thestraight chain hydrocarbons may be present as olefins; also there may beused a mixture containing straight chain parafiins and olefins.

It is an important feature of this invention that when cultivatingyeasts in the presence of the feedstock hereinbefore described underconditions favouring the growth of the yeasts at the expense of thestraight chain hydrocarbons, the other hydrocarbons, for exampleisoparafiins, naphthenes and aromatics are not metabolised or, at most,the proportion which is metabolised is very small. Furthermore, unlikeconventional chemical processes governed by the law of mass action, therate of removal of straight chain hydrocarbons is not substantiallyreduced as the proportion of these hydrocarbons in the overall mixtureof hydrocarbons decreases (except, of course, in the very final stagesof removal). Thus, when desired, the percentage conversion of straightchain hydrocarbons which is achieved can be maintained at a valueapproaching without necessitating a very disproportionate expenditure ofcontact time to achieve small improvements. Furthermore, in thecontinuous process, this high percentage conversion can be achievedwithout resorting to the use of a long reaction path.

By the. application of this process under conditions which limit themetabolisation of the straight chain hydrocarbons it is possible tooperate with the removal of only a desired proportion of thesehydrocarbons.

Suitable feedstocks to the process of the invention include kerosine,gas oils and lubricating oils; these feedstocks may be unrefined or mayhave undergone some refinery, treatment, but will usually be required tocontain a proportion of straight chain hydrocarbons in order to fulfillthe purpose of this invention. Suitably the petroleum fraction willcontain 3-45% by weight of straight chain hydrocarbons.

Microorganisms which are cultivated as herein described may be yeasts,moulds or bacteria.

Preferably when a yeast is employed this is of the familyCryptococcaceae and particularly of the sub-family Cryptococcoideae;however, if desired there may be use, for example, ascosporogeneousyeasts of the subfamily Saccharomycoideae. Preferred genera of theCryptococcoideae sub-family are Torulopsis (also known as Torula) andCandida. Preferred strains of yeast are as follows. In particular it ispreferred to use the specific stock of indicated Baarn referencenumbers; these reference numbers refer to a stock held by the CentraalBureau vor Schimmelculture, Baarn, Holland:

Candida lipolytz'ca Candida pulcherirma CBS 610 Candida utilis Candidautilis, var. major CBS 841 Candida tropicalis CBS 2317 Torulopsiscollisculosa CBS 133 3 Hansenula anomala CBS 110 Oidium lactisNeurospora sitophila Mycoderma cancoillote A Of the above Candidalipolytica is particularly preferred.

If desired, the microorganism may be a mould. A suitable strain isPenicillium expansum.

If desired, the micro-organism may be a bacterium.

Suitably the bacteria are of one of the orders: Pseudomonadales,Euba-cteriales and Actinomycetales.

Preferably the bacteria which are employed are of the family Bacillaceaeand Pseudomonadaceae. Preferred species are Bacillus megaterium,Bacillus subtilis and Pseudomonas aeruginosa. Other strains which may beemployed include:

Bacillus amylebacter Pseudomonas natricgens Arthrobacter sp. Micrococcussp. Corynebacterium sp. Psaudomonas syringae Xanthomonas begoniaeFlavobacterium devorans Acetobacter sp. Actinomyces sp.

Suitable moulds are of the family Aspergillaceae. A suitable genus isPenicillium.

Preferably there is used Penicillium expansum. Another suitable genus isAspergillus.

Usually the cultivation is carried out in the presence of an aqueousnutrient medium. If desired, certain solid nutrient media may beemployed.

In either case, a gas containing free oxygen must be provided.

Penicillium expansum is suitable for cultivation in an aqueous nutrientmedium containing hydrocarbons.

Penicillium roqueforti, Penicillium notatum Aspergil lus fussigatus andAspergillus niger, Aspergillus versicolor may be used for cultivation ona solid agent containing hydrocarbons as feedstock.

For the growth of the microorganism it will be necessary to provide, inaddition to the feedstock, an aqueous nutrient medium and a supply ofoxygen, preferably in the form of air.

A typical nutrient medium for the growth of Nocardia, a genus in theActinomycetales order, has the following composition:

Grams Ammonium sulphate 1 Magnesium sulphate 0.20 Ferrous sulphate, 7H0.005 Manganese sulphate, 1H O 0.002 Monopotassium phosphate 2 Disodiumphosphate 3 Calcium Chloride 0.1 Sodium Carbon-ate 0.1 Yeast Extract0.008 Distilled Water (to make up to 1000 mls.).

For other bacteria a suitable nutrient medium has the composition:Monopotassium phosphate gm 7 Magnesium sulphate, 7H O gm 0.2 Sodiumchloride gm 0.1 Ammonium chloride gm 2.0 Tap water (trace elements) m1100 Yeast extract gm 0.025 Made up to 1000 mls. with distilled Water.

i: A suitable nutrient medium for yeasts (and moulds) has thecomposition:

Grams Diammonium phosphate 2 Potassium chloride 1.15 Magnesium sulphate,7H O 0.65 Zinc sulphate 0.17 Manganese sulphate, 1H O 0.045 Ferroussulphate, 7H O 0.068 Tap water 200 Yeast extract 0.025

Distilled water (to make up to 1000 mls.).

The growth of the microorganism used is favoured by the addition to theculture medium of a very small proportion of extract of yeast (anindustrial product rich in vitamins of group B obtained by thehydrolysis of a yeast) or more generally of vitamins of group B and/orbiotin. This quantity is preferably of the order of 25 parts per millionwith reference to the aqueous fermentation medium. It can be higher orlower according to the conditions chosen for the growth.

The growth of the microorganism takes place at the expense of thefeedstock fraction with the intermediate production of bodies having anacid function, principally fatty acids, in such manner that the pH ofthe aqueous mineral medium progressively diminishes. If one does notcorrect it the growth is fairly rapidly arrested and the concentrationof the microorganism in the medium, that is cellular density, no longerincreases so that there is reached a so-called stationary phase.

Preferably therefore the aqueous nutrient medium is maintained at adesired pH by the step-wise or continuous addition of an aqueous mediumof high pH value. Usually, when using moulds or yeasts and in particularwhen using Candida lipolytica, the pH of the nutrient medium will bemaintained in the range 3-6 and preferably in the range 4-5. (Bacteriarequire a higher pH, usually 6.5-8.) Suitable alkaline materials foraddition to the growth mixture include sodium hydroxide, potassiumhydroxide, d-isodium hydrogen phosphate and ammonia, either free or inaqueous solution.

The optimum temperature of the growth mixture will vary according to thetype of microorganism employed and will usually lie in the range 25-35C. When using Candida lipolytica the preferred temperature range is28-32 C.

The take-up of oxygen is essential for the growth of the microorganism.The oxygen will usually be provided as air. In order to maintain a rapidrate of growth the air, used to provide oxygen, should be present in theform of fine bubbles under the action of stirring. The air may beintroduced through a sintered surface. However there may be used thesystem of intimate aeration known as vortex aeration.

It has been found that by the use of yeast of the strain Candidalipolytica in a process according to the invention in which aeration iseffected by vortex aeration, a high growth rate is achieved whereby thegeneration time lies in the range 2-5 hours and the cell concentrationis increased by a factor of up to 12 in two days.

In batch operation, the microorganism will usually grow initially at alow rate of increase in cellular density. (This period of growth isreferred to as the lag phase.) Subsequently the rate of growth willincrease to a higher rate of growth; the period at the higher rate ofgrowth is referred to as the exponential phase and subsequently againthe cellular density will become constant (the stationary phase).

A supply of the microorganism for starting the next batch willpreferably be removed before the termination of the exponential phase.

The growth operation will usually be discontinued before the stationaryphase.

At this stage, the microorganism will usually be separated from the bulkof the aqueous nutrient medium and from the bulk of the un-usedfeedstock fraction.

If desired the microorganism may be subjected to autolysis beforefurther purification of the product.

According to one method of treating the product the major part of thecontinuous aqueous phase is first separated; preferably this is carriedout by centrifuging, or decanting. The separated aqueous phase willusually contain :a greater concentration of non-nutritive ions than canbe tolerated in the recycle stream and when this is so, only aproportion of the recovered aqueous phase can be recycled. Thus it willusually be possible to separate ca. 96% by wt. of the aqueous phasewhich is present in the product, of which on the same percentage basis,ca. 20% by wt. will be discarded. The recycle stream is supplied withmake-up quantities of the necessary nutrients and is returned to thefermenter; if desired the make-up materials may be fed to the fermenteras a separate stream.

The process, as applied to the cultivation of a yeast, may incorporateproduct separation stages as follows. In some cases microorganisms otherthan yeasts may be separated in this manner.

By centrifuging the product from the fermenter three fractions arerecovered. These are in order of increasing density:

(i) an oil phase containing yeast cells (ii) an aqueous phase containingtraces of oil and yeast,

and

(iii) a yeast cream consisting of yeast, having a quantity of oil fixedonto the cells, together with aqueous phase.

After recovery of fraction (ii), fraction (iii) or a blend of fractions(i) and (iii) is mixed with an aqueous solution of a surfactant.

The purpose of this treatment is to separate at least part of the oilfrom the yeast cells; the oil being apparently held to the cells byadsorption.

It may be advantageous to employ an edible surfactant, for example asaccharose ester, which makes it possible to reduce the subsequentWashing required to remove from the yeast a surfactant which is notedible.

The emulsion so formed is broken down by centrifuging to obtain threefractions:

(iv) an oil phase (v) an aqueous phase containing surfactant, whichphase is recycled for the treatment of fractions (i) and (iii), and

(vi) a yeast cream, consisting of yeast still contaminated by oiltogether with an aqueous surfactant phase.

In order to reduce as far as possible the consumption of surfactantproduct, the aqueous washing solution containing it is recycled.

Fraction (vi) may be further treated by alternate washing withsurfactant and centrifuging until the oil content of the yeast hasreached a desired low value. The yeast cream now consisting of yeast andaqueous surfactant may now be washed with water and again centrifuged.If desired two or more washings may be given to this yeast cream. Ifdesired, one or more of these water washings (but preferably not thelast) may make use of salt water (for example sea water); preferably thefinal wash is with soft water. With a view to economising the soft waternecessary for the process, the whole of this water coming from the lastwashing is employed for making up the nutritive medium for thefermentation, where necessary at the stage of washing with the solutionof surfactant, and the rest is sent to the salt water used for washingwith a view to reducing its salt concentration. Finally the yeast may bedried under conditions suitable for its subsequent use as a foodstuff.

Other steps which may be taken to obtain a purified microorganism or aproduct derived therefrom or to improve the process in respect of theproduction of the unmetabolised hydrocarbon fraction are described inthe following applications; the use of any process step or steps thereindescribed in association with the process herein described lies withinthe scope of the present invention.

The stages of the process may be carried out entirely batchwise.However, if desired, any one or more stages herein described may becarried out in continuous manner.

The invention is illustrated but not limited with reference to thefollowing experiment and example.

Throughout these examples cellular density is expressed as dry weight ofyeast per litre of culture.

The experiment is provided for purposes of comparison and does notconstitute operation according to the present invention.

Experiment 40 litres of an aqueous mineral nutrient medium wereintroduced into a stainless steel fermenter having an effective capacityof 60 litres.

20 litres of an inoculum of 24 hr. Candida lipolytica culture on normalparafiinic hydrocarbons (in the stationary growth phase, cellulardensity 5 grams/litre) was then introduced into the fermenter, giving acellular density of about 1 gram/litre in the fermenter.

1.03 litres, that is 15 grams/litre, of heavy gas-oil was then added tothe fermenter, that is sufiicient to take the cellular density to 2grams/litre.

The temperature of the culture was regulated at 30i1 C., pH 4, aerationand agitation to give 3 millimoles of 0 per litre of medium per minute.An automatic pH controller added 10 N ammonia as required.

When the flow of ammonia reached 20 ml. the addition of gas-oil wasstarted following the theoretical needs of the culture, assuming a yielddry yeast X gas-oil required of 10% and a cell division time of 3 hours.This addition was carried out every hour until a total of 250grams/litre of gas-oil had been added, Le. 17 litres.

The time taken for this addition was 15 hours. Starting with a densityof 1 gram/litre a lag period of 5 hours followed; then an accelerationphase of 3 hours and finally a phase of exponential growth whichcontinues to 18 grams/ litre at 22 hours.

Example The method described in the experiment was repeated but using,an inoculum, 20 litres of a 24 hr. Candida lipolytica culture onmolasses (cellular density 6 grams/ litre in the stationary growthphase). The inoculum was prepared using 30 grams/litre of beet molasses,containing among other growth factors 0.04 mg./kg. of biotin, 50 mg./kg.of pantothenic acid and 5 mg./kg. of inositol, these three factors beingparticularly valuable for the growth of yeasts. When the inoculum wasemployed on the heavy gas-oil there was no lag phase, the culturestarting off immediately in the phase of maximum growth (cell divisiontime 3 hours).

The aqueous nutrient. medium used in the process of the experiment andexample had the following composition:

Grams Diammonium phosphate 2 Potassium chloride 1.15 Magnesium sulphate,7H O 0.65 Zinc sulphate 0.17 Manganese sulphate, IH O 0.045 Ferroussulphate, 7H O 0.068 Yeast extract 0.025 Tap water 200 Distilled waterto 1000 ml.

We claim:

1. A process which comprises, in a pretreatment stage, cultivating acarbohydrate consuming yeast in the presence of a pretreatment feedstockcomprising a carbohydrate and thereafter, in a growth stage, cultivatingthe yeast so obtained in the presence of a growth feedstock comprising astraight chain hydrocarbon.

2. A process according to claim 1 in which the carbohydrate is containedin a class of material selected from the group consisting of molasses,wort, malt extract, wood hydrolysis sugars and lyes obtained in thecourse of paper manufacture.

3. A process according to claim 1 in which the yeast is of the familyCryptococcaceae.

4. A process according to claim 3 in which the yeast is of thesub-family Cryptococcoideae.

5. A process according to claim 4 in which the yeast is of the genusTorulopsis.

6. A process according to claim 4 in which the yeast is of the genusCandida.

7. A process according to claim 4 in which the yeast is Candidalipolytica.

8. A process according to claim 1 in which the feedstock is a petroleumfraction.

9. A process according to claim 1 in which straight chain hydrocarbonsare removed from a petroleum fraction With production of a yeast,wherein the feedstock employed for the growth of the micro-organism is apetroleum fraction consisting in part of straight chain hydrocarbons,whereby there is recovered from the product of the growth of the yeast apetroleum fraction having a reduced proportion of straight chainhydrocarbons or which is free of said straight chain hydrocarbons.

10. A process according to claim 9 for the removal, at least in part, ofwaxes from a wax-containing petroleum gas oil, wherein the feedstock isa wax-containing petroleum gas oil and wherein there is recovered, fromthe product'of the growth of the microorganism, a gas oil of reducedcontent of wax.

References Cited by the Examiner OTHER REFERENCES Cook, The Chemistryand Biology of Yeasts, Academic Press Inc., New York, 8, pages 648-659.

Wickerham et al., Carbon Assimilation Tests for the Classification ofYeasts, Journal of Bacteriology 56, 1948, pages 363-371.

A. LOUIS MONACELL, Primary Examiner.

D. M. STEPHENS, Assistant Examiner.

1. A PROCESS WHICH COMPRISES, IN A PRETREATMENT STAGE, CULTIVATING A CARBOHYDRATE CONSUMING YEAST IN THE PRESENCE OF A PRETREATMENT FEEDSTOCK COMPRISING A CARBOHYDRAGE AND THEREAFTER, IN A GROWTH STAGE, CULTIVATING THE YEAST SO OBTAINED IN THE PRESENCE OF A GROWTH FEEDSTOCK COMPRISING A STRAIGHT CHAIN HYDROCARGON. 